Methods of oil production in microorganisms

ABSTRACT

Provided herein are methods for producing one or more polyunsaturated fatty acids. The methods include the steps of providing a microorganism capable of producing polyunsaturated fatty acids, providing a medium comprising a high concentration of one or more carbon sources, low pH, or both, and culturing the microorganism in the medium under sufficient conditions to produce the one or more polyunsaturated fatty acids. Also provided are methods of culturing one or more microorganisms. The methods include culturing the microorganisms in a medium comprising a first amount of one or more carbon sources at a first concentration level, monitoring a carbon source concentration until the carbon source concentration is reduced below the first concentration level, and adding to the medium a second amount of one or more carbon sources to increase the carbon source concentration to a second concentration level.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Application No.62/001,912, filed May 22, 2014, which is incorporated herein byreference in its entirety.

BACKGROUND

In the field of oil production via fermentation of eukaryoticmicroorganisms, certain strategies have been established and widelyaccepted. One such strategy to avoid nutrient inhibition and to achievehigh cell concentration is using fed-batch fermentation, in which themain substrates (mainly the carbon sources) are added in increments tomaintain a continuous supply while avoiding high concentrations of thesubstrates in the fermentation medium. However, fed-batch fermentationusually requires careful planning of the substrate feeding regime andintensive real-time fermentation monitoring and control, which demandsextensive man power and may lead to a high failure rate of thefermentation operation.

Another significant cost related to fermentation on an industrial scaleincludes procedures related to sterilization. These costs includeexpensive pressure vessel fermenters and steam-in-place systems as wellas the associated operating costs for generating the steam.

SUMMARY

Provided herein are methods for producing one or more polyunsaturatedfatty acids. The methods include the steps of providing a microorganismcapable of producing polyunsaturated fatty acids, providing a mediumcomprising a high concentration of one or more carbon sources, low pH,or both, and culturing the microorganism in the medium under sufficientconditions to produce the one or more polyunsaturated fatty acids.

Also provided are methods of reducing contamination of a non-sterileculture of one or more microorganisms. The methods include culturing themicroorganisms (i) in the presence of a high concentration of one ormore carbon sources, (ii) under conditions of low pH, or (iii) acombination thereof, wherein the culturing reduces contamination of thenon-sterile culture comprising the microorganisms.

Provided are methods of culturing one or more microorganisms. Themethods include culturing the microorganisms in a medium comprising afirst amount of one or more carbon sources at a first concentrationlevel, monitoring a carbon source concentration until the carbon sourceconcentration is reduced below the first concentration level, and addingto the medium a second amount of one or more carbon sources to increasethe carbon source concentration to a second concentration level.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the time profile of ONC-T18 cell concentration(biomass (“X”)) and total fatty acid content (TFA %) during a 2 liter(L) fermentation.

FIG. 2 is a graph showing the time profile of ONC-T18 cell concentration(biomass (“X”)) and total fatty acid content (TFA %) during a 5 Lfermentation.

FIG. 3 is a graph showing the time profile of ONC-T18 cell concentration(biomass (“X”)) and total fatty acid content (TFA %) during a 30 Lfermentation.

FIG. 4 is a graph showing the time profile of ONC-T18 and ATCC20888 cellconcentration during parallel high glucose fermentations using the samefermentation medium formula (ONC formula).

FIG. 5 is a graph showing the time profile of ONC-T18 and ATCC20888 cellconcentration during parallel high glucose fermentations using the ONCfermentation medium formula for ONC-T18 and a different fermentationmedium formula for ATCC20888.

FIG. 6 is a graph showing the time profile of ONC-T18 cell concentration(biomass (“X”)) and glucose concentration during a 30 L high glucosemulti-batch fermentation. FIG. 6 and FIG. 3 show data from the same 30 Lfermentation.

FIG. 7 is a graph showing the effect of pH on the growth of ONC-T18under high glucose multi-batch fermentation conditions.

FIG. 8 is a graph showing the fatty acid profiles of ONC-T18 grown underdifferent pH conditions and using high glucose multi-batch fermentation.

FIG. 9 is a graph showing reduced contamination of an ONC-T18 cultureunder low pH conditions.

DETAILED DESCRIPTION

Provided herein are methods for producing one or more polyunsaturatedfatty acids. The methods include providing a microorganism capable ofproducing polyunsaturated fatty acids, providing a medium comprising ahigh concentration of one or more carbon sources, low pH, or both, andculturing the microorganism in the medium under sufficient conditions toproduce the one or more polyunsaturated fatty acids. Optionally, themedium has a low pH. Optionally, the medium has a high concentration ofone or more carbon sources. Optionally, the medium has a low pH and ahigh concentration of one or more carbon sources.

Also provided herein is a method of reducing contamination of anon-sterile culture comprising one or more microorganisms. The methodincludes culturing the microorganisms (i) in the presence of a highconcentration of one or more carbon sources, (ii) under conditions oflow pH, or (iii) a combination thereof, wherein the culturing reducescontamination of the non-sterile culture comprising the microorganisms.Optionally, the method comprises culturing the microorganisms in an openvessel. Optionally, the culturing comprises culturing the microorganismsin the presence of a high concentration of one or more carbon sources.Optionally, the culturing comprises culturing the microorganisms underconditions of low pH. Optionally, the culturing comprises culturing themicroorganisms in the presence of a high concentration of one or morecarbon sources and under conditions of low pH.

As used herein, the term “low pH” or “reduced pH” refers to a pH valuelower than neutral pH. The term “low pH” generally refers to a pH valuelower than 4.5. Optionally, low pH refers to a value of 2 to 4.5,inclusive, or any value between 2 and 4.5. Optionally, the pH is 3 to3.5. It is understood that the pH of a culture may change over time,i.e., over the course of the fermentation process. As used herein,culturing the microorganism under conditions of low pH means that the pHof the culture or medium is monitored and adjusted over time to maintainthe pH of the culture at low pH.

As used herein, the phrase “high concentration of a carbon source”refers to an amount of the carbon source of at least 200 g/L. Forexample, the concentration of the one or more carbon sources can be atleast 200 g/L or greater than 200 g/L. Optionally, the concentration ofthe one or more carbon sources is 200 to 300 g/L. Optionally, theconcentration of the one or more carbon sources is 200 to 250 g/L. It isunderstood that the concentration of a carbon source may change overtime, i.e., over the course of the fermentation process. As used herein,a medium containing a high concentration of a carbon source means thatthe medium contains at least 200 g/L of the carbon source. As usedherein, culturing the microorganism in a high concentration of a carbonsource means that the initial concentration of the carbon source in theculture or medium is at least 200 g/L. As described in more detailbelow, the carbon source concentration can be monitored over time one ormore times and once it reaches a certain threshold an additional amountof a carbon source can be added to the culture or medium. In thisinstance, the additional amount of the carbon source is a highconcentration of a carbon source, i.e., at least 200 g/L of the carbonsource.

Thus, provided is a method of culturing one or more microorganisms. Themethods include culturing the microorganisms in a medium comprising afirst amount of one or more carbon sources at a first concentrationlevel, monitoring a carbon source concentration until the carbon sourceconcentration is reduced below the first concentration level, and addingto the medium a second amount of one or more carbon sources to increasethe carbon source concentration to a second concentration level.Optionally, the first and/or second concentration levels of the one ormore carbon sources are greater than 200 g/L. Optionally, the secondamount of the one or more carbon sources is added to the medium when thecarbon source concentration level is reduced to 0 to 20 g/L. Theprovided methods can include repeated rounds of monitoring and additionsof carbon sources as desired. Thus, the provided methods can include,after addition of the second amount of the one or more carbon sources,(a) culturing the microorganisms until the carbon source concentrationof the one or more carbon sources is reduced below the secondconcentration level and (b) adding to the medium a third amount of oneor more carbon sources to increase the carbon source concentration to athird concentration level. Optionally, the third concentration level ofthe one or more carbon sources is greater than 200 g/L. Optionally, thethird amount of the one or more carbon sources is added to the mediumwhen the carbon source concentration is reduced to 0 to 20 g/L.Optionally, the methods include, after addition of the third amount ofthe one or more carbon sources, (a) culturing the microorganisms untilthe carbon source concentration of the one or more carbon sources isreduced below the third concentration level and (b) adding to the mediuma fourth amount of one or more carbon sources to increase the carbonsource concentration to a fourth concentration level. Optionally, thefourth concentration level of the one or more carbon sources is greaterthan 200 g/L. Optionally, the fourth amount of the one or more carbonsources is added to the medium when the carbon source concentration isreduced to 0 to 20 g/L. Optionally, the one or more carbon sources inthe first, second, third, and fourth amounts are the same.

In the provided methods, the carbon source concentration can bemonitored one or more times. Optionally, the carbon source concentrationcan be monitored continuously (e.g., using a device that continuouslymonitors carbon source (e.g., glucose) concentrations in a medium) orperiodically (e.g., by removing a sample of medium and testing carbonsource concentration in the sample). Optionally, the carbon sourceconcentration is monitored or determined before and/or after addition ofan amount of the one or more carbon sources. Thus, for example, theprovided methods can include monitoring the carbon source concentrationone or more times between additions of the amounts of the one or morecarbon sources. Optionally, the provided methods include monitoring ordetermining the carbon source concentration before addition of an amountof one or more carbon sources, after addition of an amount of one ormore carbon sources and one or more times prior to addition of a furtheramount of one or more carbon sources. By way of example, the providedmethods can include monitoring the carbon source concentration afteraddition of a first amount of the one or more carbon sources and,optionally, one or more times prior to addition of a second amount ofthe one or more carbon sources. The provided methods can includemonitoring the carbon source concentration after addition of the secondamount of the one or more carbon sources and, optionally, one or moretimes prior to addition of a third amount of the one or more carbonsources. The provided methods can include monitoring the carbon sourceconcentration after addition of the third amount of the one or morecarbon sources and, optionally, one or more times prior to addition of afourth amount of the one or more carbon sources. Optionally, in theprovided methods, the carbon source concentration is monitored oncebetween each addition of the amounts of the one or more carbon sources.By way of example, the carbon source concentration is monitored afteraddition of the first amount of the one or more carbon sources and priorto addition of the second amount of the one or more carbon sources onetime. Similarly, the carbon source concentration can be monitored afteraddition of the second amount of the one or more carbon sources andprior to addition of the third amount of the one or more carbon sourcesone time. Optionally, the carbon source concentration is monitored onetime prior to addition of the second amount of the one or more carbonsources regardless of the number of further additions of amounts of theone or more carbon sources.

Carbon source concentration or levels can be monitored directly orindirectly by any means known to those of skill in the art. Optionally,the carbon source concentration is monitored by measuring dissolvedoxygen levels, e.g., in the medium or in a sample from the medium.Optionally, the monitoring includes obtaining a sample of the medium anddetermining the carbon source concentration in the sample. Optionally,the step of determining the carbon source concentration comprises acolorimetric, enzyme-based, or fluorescence assay. Optionally, the stepof determining carbon source concentration includes high pressure liquidchromatograph (HPLC).

I. Microorganisms

The methods described herein include extracting lipids from a populationof microorganisms. The population of microorganisms described herein canbe algae (e.g., microalgae), fungi (including yeast), bacteria, orprotists. Optionally, the microorganism includes Thraustochytrids of theorder Thraustochytriales, more specifically Thraustochytriales of thegenus Thraustochytrium. Optionally, the population of microorganismsincludes Thraustochytriales as described in U.S. Pat. Nos. 5,340,594 and5,340,742, which are incorporated herein by reference in theirentireties. The microorganism can be a Thraustochytrium species, such asthe Thraustochytrium species deposited as ATCC Accession No. PTA-6245(i.e., ONC-T18) as described in U.S. Pat. No. 8,163,515, which isincorporated by reference herein in its entirety. Thus, themicroorganism can have an 18s rRNA sequence that is at least 95%, 96%,97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%,99.9% or more (e.g., including 100%) identical to SEQ ID NO:1.

The microorganisms for use in the methods described herein can produce avariety of lipid compounds. As used herein, the term lipid includesphospholipids, free fatty acids, esters of fatty acids,triacylglycerols, sterols and sterol esters, carotenoids, xanthophyll(e.g., oxycarotenoids), hydrocarbons, and other lipids known to one ofordinary skill in the art. Optionally, the lipid compounds includeunsaturated lipids. The unsaturated lipids can include polyunsaturatedlipids (i.e., lipids containing at least 2 unsaturated carbon-carbonbonds, e.g., double bonds) or highly unsaturated lipids (i.e., lipidscontaining 4 or more unsaturated carbon-carbon bonds). Examples ofunsaturated lipids include omega-3 and/or omega-6 polyunsaturated fattyacids, such as docosahexaenoic acid (i.e., DHA), eicosapentaenoic acid(i.e., EPA), and other naturally occurring unsaturated, polyunsaturatedand highly unsaturated compounds.

II. Processes

Fermentation

The provided methods include or can be used in conjunction withadditional steps for culturing microorganisms according to methods knownin the art. For example, a Thraustochytrid, e.g., a Thraustochytriumsp., can be cultivated according to methods described in U.S. PatentPublication US 2009/0117194 or US 2012/0244584, which are hereinincorporated by reference in their entireties. Microorganisms are grownin a growth medium (also known as “culture medium”). Any of a variety ofmedium can be suitable for use in culturing the microorganisms describedherein. Optionally, the medium supplies various nutritional components,including a carbon source and a nitrogen source, for the microorganism.

Medium for Thraustochytrid culture can include any of a variety ofcarbon sources. Examples of carbon sources include fatty acids, lipids,glycerols, triglycerols, carbohydrates, polyols, amino sugars, and anykind of biomass or waste stream. Fatty acids include, for example, oleicacid. Carbohydrates include, but are not limited to, glucose,celluloses, hemicelluloses, fructose, dextrose, xylose, lactulose,galactose, maltotriose, maltose, lactose, glycogen, gelatin, starch(corn or wheat), acetate, m-inositol (e.g., derived from corn steepliquor), galacturonic acid (e.g., derived from pectin), L-fucose (e.g.,derived from galactose), gentiobiose, glucosamine,alpha-D-glucose-1-phosphate (e.g., derived from glucose), cellobiose,dextrin, alpha-cyclodextrin (e.g., derived from starch), and sucrose(e.g., from molasses). Polyols include, but are not limited to,maltitol, erythritol, and adonitol. Amino sugars include, but are notlimited to, N-acetyl-D-galactosamine, N-acetyl-D-glucosamine, andN-acetyl-beta-D-mannosamine. Optionally, the carbon source is glucose.As noted above, in the provided methods, the carbon source is providedat a high concentration, e.g., at least 200 g/L.

Optionally, the microorganisms provided herein are cultivated underconditions that increase biomass and/or production of a compound ofinterest (e.g., oil or total fatty acid (TFA) content).Thraustochytrids, for example, are typically cultured in saline medium.Optionally, Thraustochytrids can be cultured in medium having a saltconcentration from about 2.0 g/L to about 50.0 g/L. Optionally,Thraustochytrids are cultured in medium having a salt concentration fromabout 2 g/L to about 35 g/L (e.g., from about 18 g/L to about 35 g/L).Optionally, the Thraustochytrids described herein can be grown in lowsalt conditions. For example, the Thraustochytrids can be cultured in amedium having a salt concentration from about 5 g/L to about 20 g/L(e.g., from about 5 g/L to about 15 g/L). The culture medium optionallyinclude NaCl. Optionally, the medium include natural or artificial seasalt and/or artificial seawater.

The culture medium can include non-chloride-containing sodium salts(e.g., sodium sulfate) as a source of sodium. For example, a significantportion of the total sodium can be supplied by non-chloride salts suchthat less than about 100%, 75%, 50%, or 25% of the total sodium inculture medium is supplied by sodium chloride.

Optionally, the culture medium have chloride concentrations of less thanabout 3 g/L, 500 mg/L, 250 mg/L, or 120 mg/L. For example, culturemedium for use in the provided methods can have chloride concentrationsof between and including about 60 mg/L and 120 mg/L.

Examples of non-chloride sodium salts suitable for use in accordancewith the present methods include, but are not limited to, soda ash (amixture of sodium carbonate and sodium oxide), sodium carbonate, sodiumbicarbonate, sodium sulfate, and mixtures thereof. See, e.g., U.S. Pat.Nos. 5,340,742 and 6,607,900, the entire contents of each of which areincorporated by reference herein.

Medium for Thraustochytrids culture can include any of a variety ofnitrogen sources. Exemplary nitrogen sources include ammonium solutions(e.g., NH₄ in H₂O), ammonium or amine salts (e.g., (NH₄)₂SO₄, (NH₄)₃PO₄,NH₄NO₃, NH₄OOCH₂CH₃ (NH₄Ac)), peptone, tryptone, yeast extract, maltextract, fish meal, sodium glutamate, soy extract, casamino acids anddistiller grains. Concentrations of nitrogen sources in suitable mediumtypically range between and including about 1 g/L and about 25 g/L.

The medium optionally include a phosphate, such as potassium phosphateor sodium-phosphate. Inorganic salts and trace nutrients in medium caninclude ammonium sulfate, sodium bicarbonate, sodium orthovanadate,potassium chromate, sodium molybdate, selenous acid, nickel sulfate,copper sulfate, zinc sulfate, cobalt chloride, iron chloride, manganesechloride calcium chloride, and EDTA. Vitamins such as pyridoxinehydrochloride, thiamine hydrochloride, calcium pantothenate,p-aminobenzoic acid, riboflavin, nicotinic acid, biotin, folic acid andvitamin B12 can be included.

The pH of the medium can be adjusted to between and including 3.0 and10.0 using acid or base, where appropriate, and/or using the nitrogensource. Optionally, the medium is adjusted to a low pH as defined above.The medium can be sterilized.

Generally a medium used for culture of a microorganism is a liquidmedium. However, the medium used for culture of a microorganism can be asolid medium. In addition to carbon and nitrogen sources as discussedherein, a solid medium can contain one or more components (e.g., agar oragarose) that provide structural support and/or allow the medium to bein solid form.

Cells can be cultivated for anywhere from 1 day to 60 days. Optionally,cultivation is carried out for 14 days or less, 13 days or less, 12 daysor less, 11 days or less, 10 days or less, 9 days or less, 8 days orless, 7 days or less, 6 days or less, 5 days or less, 4 days or less, 3days or less, 2 days or less, or 1 day or less. Cultivation isoptionally carried out at temperatures from about 4° C. to about 30° C.,e.g., from about 18° C. to about 28° C. Cultivation can includeaeration-shaking culture, shaking culture, stationary culture, batchculture, semi-continuous culture, continuous culture, rolling batchculture, wave culture, or the like. Cultivation can be performed using aconventional agitation-fermenter, a bubble column fermenter (batch orcontinuous cultures), a wave fermenter, etc.

Cultures can be aerated by one or more of a variety of methods,including shaking. Optionally, shaking ranges from about 100 rpm toabout 1000 rpm, e.g., from about 350 rpm to about 600 rpm or from about100 to about 450 rpm. Optionally, the cultures are aerated usingdifferent shaking speeds during biomass-producing phases and duringlipid-producing phases. Alternatively or additionally, shaking speedscan vary depending on the type of culture vessel (e.g., shape or size offlask).

Optionally, the level of dissolved oxygen (DO) is higher during thebiomass production phase than it is during the lipid production phase.Thus, DO levels are reduced during the lipid production phase (i.e., theDO levels are less than the amount of dissolved oxygen in biomassproduction phase). Optionally, the level of dissolved oxygen is reducedbelow saturation. For example, the level of dissolved oxygen can bereduced to a very low, or even undetectable, level.

The production of desirable lipids can be enhanced by culturing cellsaccording to methods that involve a shift of one or more cultureconditions in order to obtain higher quantities of desirable compounds.Optionally, cells are cultured first under conditions that maximizebiomass, followed by a shift of one or more culture conditions toconditions that favor lipid productivity. Conditions that are shiftedcan include oxygen concentration, C:N ratio, temperature, andcombinations thereof. Optionally, a two-stage culture is performed inwhich a first stage favors biomass production (e.g., using conditions ofhigh oxygen (e.g., generally or relative to the second stage), low C:Nratio, and ambient temperature), followed by a second stage that favorslipid production (e.g., in which oxygen is decreased, C:N ratio isincreased, and temperature is decreased).

Pasteurization

Optionally, the resulting biomass is pasteurized to inactivateundesirable substances present in the biomass. For example, the biomasscan be pasteurized to inactivate compound degrading substances. Thebiomass can be present in the fermentation medium or isolated from thefermentation medium for the pasteurization step. The pasteurization stepcan be performed by heating the biomass and/or fermentation medium to anelevated temperature. For example, the biomass and/or fermentationmedium can be heated to a temperature from about and including 50° C. toabout and including 95° C. (e.g., from about and including 55° C. toabout and including 90° C. or from about and including 65° C. to aboutand including 80° C.). Optionally, the biomass and/or fermentationmedium can be heated from about and including 30 minutes to about andincluding 120 minutes (e.g., from about and including 45 minutes toabout and including 90 minutes, or from about and including 55 minutesto about and including 75 minutes). The pasteurization can be performedusing a suitable heating means as known to those of skill in the art,such as by direct steam injection.

Optionally, a pasteurization step is not performed (i.e., the methodlacks a pasteurization step.

Harvesting and Washing

Optionally, the biomass can be harvested according to methods known tothose of skill in the art. For example, the biomass can optionally becollected from the fermentation medium using various conventionalmethods, such as centrifugation (e.g., solid-ejecting centrifuges) orfiltration (e.g., cross-flow filtration) and can also include the use ofa precipitation agent for the accelerated collection of cellular biomass(e.g., sodium phosphate or calcium chloride).

Optionally, the biomass is washed with water. Optionally, the biomasscan be concentrated up to about and including 20% solids. For example,the biomass can be concentrated to about and including 5% to about andincluding 20% solids, from about and including 7.5% to about andincluding 15% solids, or from about and including 15% solids to aboutand including 20% solids, or any percentage within the recited ranges.Optionally, the biomass can be concentrated to about 20% solids or less,about 19% solids or less, about 18% solids or less, about 17% solids orless, about 16% solids or less, about 15% solids or less, about 14%solids or less, about 13% solids or less, about 12% solids or less,about 11% solids or less, about 10% solids or less, about 9% solids orless, about 8% solids or less, about 7% solids or less, about 6% solidsor less, about 5% solids or less, about 4% solids or less, about 3%solids or less, about 2% solids or less, or about 1% solids or less.

Isolation and Extraction

The provided methods, optionally, include isolating the polyunsaturatedfatty acids from the biomass or microorganisms using methods known tothose of skill in the art. For example, methods of isolatingpolyunsaturated fatty acids are described in U.S. Pat. No. 8,163,515,which is incorporated by reference herein in its entirety. Optionally,the medium is not sterilized prior to isolation of the polyunsaturatedfatty acids. Optionally, sterilization comprises an increase intemperature. Optionally, the polyunsaturated fatty acids produced by themicroorganisms and isolated from the provided methods are medium chainfatty acids. Optionally, the one or more polyunsaturated fatty acids areselected from the group consisting of alpha linolenic acid, arachidonicacid, docosahexanenoic acid, docosapentaenoic acid, eicosapentaenoicacid, gamma-linolenic acid, linoleic acid, linolenic acid, andcombinations thereof.

III. Products

Polyunsaturated fatty acids (PUFAs) and other lipids produced accordingto the method described herein can be utilized in any of a variety ofapplications, for example, exploiting their biological or nutritionalproperties. Optionally, the compounds can be used in pharmaceuticals,food supplements, animal feed additives, cosmetics, and the like.Optionally, the PUFAs and other lipids are used to produce fuel, e.g.,biofuel. Lipids produced according to the methods described herein canalso be used as intermediates in the production of other compounds.

Optionally, the lipids produced according to the methods describedherein can be incorporated into a final product (e.g., a food or feedsupplement, an infant formula, a pharmaceutical, a fuel, etc.) Suitablefood or feed supplements for incorporating the lipids described hereininto include beverages such as milk, water, sports drinks, energydrinks, teas, and juices; confections such as jellies and biscuits;fat-containing foods and beverages such as dairy products; processedfood products such as soft rice (or porridge); infant formulae;breakfast cereals; or the like. Optionally, one or more produced lipidscan be incorporated into a dietary supplement, such as, for example, amultivitamin. Optionally, a lipid produced according to the methoddescribed herein can be included in a dietary supplement and optionallycan be directly incorporated into a component of food or feed (e.g., afood supplement).

Examples of feedstuffs into which lipids produced by the methodsdescribed herein can be incorporated include pet foods such as catfoods; dog foods and the like; feeds for aquarium fish, cultured fish orcrustaceans, etc.; feed for farm-raised animals (including livestock andfish or crustaceans raised in aquaculture). Food or feed material intowhich the lipids produced according to the methods described herein canbe incorporated is preferably palatable to the organism which is theintended recipient. This food or feed material can have any physicalproperties currently known for a food material (e.g., solid, liquid,soft).

Optionally, one or more of the produced compounds (e.g., PUFA) can beincorporated into a pharmaceutical. Examples of such pharmaceuticalsinclude various types of tablets, capsules, drinkable agents, etc.Optionally, the pharmaceutical is suitable for topical application.Dosage forms can include, for example, capsules, oils, granula, granulasubtilae, pulveres, tabellae, pilulae, trochisci, or the like.

The lipids produced according to the methods described herein can beincorporated into products as described herein by combinations with anyof a variety of agents. For instance, such compounds can be combinedwith one or more binders or fillers. In some embodiments, products caninclude one or more chelating agents, pigments, salts, surfactants,moisturizers, viscosity modifiers, thickeners, emollients, fragrances,preservatives, etc., and combinations thereof.

Disclosed are materials, compositions, and components that can be usedfor, can be used in conjunction with, can be used in preparation for, orare products of the disclosed methods and compositions. These and othermaterials are disclosed herein, and it is understood that whencombinations, subsets, interactions, groups, etc. of these materials aredisclosed that while specific reference of each various individual andcollective combinations and permutations of these compounds may not beexplicitly disclosed, each is specifically contemplated and describedherein. For example, if a method is disclosed and discussed and a numberof modifications that can be made to a number of molecules including themethod are discussed, each and every combination and permutation of themethod, and the modifications that are possible are specificallycontemplated unless specifically indicated to the contrary. Likewise,any subset or combination of these is also specifically contemplated anddisclosed. This concept applies to all aspects of this disclosureincluding, but not limited to, steps in methods using the disclosedcompositions. Thus, if there are a variety of additional steps that canbe performed, it is understood that each of these additional steps canbe performed with any specific method steps or combination of methodsteps of the disclosed methods, and that each such combination or subsetof combinations is specifically contemplated and should be considereddisclosed.

Publications cited herein and the material for which they are cited arehereby specifically incorporated by reference in their entireties.

The examples below are intended to further illustrate certain aspects ofthe methods and compositions described herein, and are not intended tolimit the scope of the claims.

EXAMPLES Example 1 High Glucose Multi-Batch Fermentation ofMicroorganisms for Oil Production

During our fermentation process development based on ONC-T18, theculture could survive extremely high concentration of glucose in thefermentation medium (up to 250 g/L). Such observation has led todevelopment of fermentation processes with intentionally high initialglucose concentration and high glucose doses during the fermentation.

Thus, as described herein, unique oil fermentation processes formicroorganisms was developed. Strategies including high initialconcentration (up to 250 g/L) of glucose in the medium and high dosesglucose supply during the fermentation were employed to achieve fastculture growth and oil production. Such carbon source supply strategyprovides simpler yet highly efficient alternative to the continuouscarbon feeding strategies employed by traditional fed-batch fermentationprocesses. The potential of foreign organism contamination was greatlyreduced due to high osmotic pressure created by the high carbonsubstrate concentration. Such fermentation strategy was also applied tofermentations of a representative algal oil production strain,Schizochytrium sp. ATCC20888. However, no significant culture growthcould be achieved under high glucose conditions. It was thereforedemonstrated that it may be a unique trait of ONC-T18 as well as someother microorganisms in coping with such high concentration of carbonsource.

High glucose fermentations of ONC-T18 were carried out at differentfermentor scales with the same medium composition and glucose supplystrategy. The fermentors used were 2 liter (L), 5 L, and 30 L withworking volume of about 1.7 L, 4 L, and 25 L, respectively. Mediumcomposition and glucose supply strategy are detailed in Table 1 below.

TABLE 1 Medium composition and glucose supply strategy during highglucose multi-batch fermentations of ONC-T18 2 L 5 L 30 L fermentorfermentor fermentor Initial 1.3 L 3.5 L 25 L volume Final 1.7 L 4.5 L 25L volume Initial Glucose 242 g/L 230 g/L 204 g/L medium Soy peptone 2g/L 2 g/L 2 g/L MgSO4•7H2O 4 g/L 4 g/L 4 g/L FeCl3•6H2O 0.005 g/L 0.005g/L 0.005 g/L Trace elements 1.5 ml/L 1.5 ml/L 1.5 ml/L solution (stock)KH2PO4 2.2 g/L 2.2 g/L 2.2 g/L K2HPO4 2.4 g/L 2.4 g/L 2.4 g/L (NH4)2SO420 g/L 20 g/L 20 g/L Vitamin 3 g/L 3 g/L 3 g/L solution (stock)CaCl2•2H2O 0.1 g/L 0.1 g/L 0.1 g/L Base 5M NaOH As needed solution Acid2M H2SO4 As needed solution Glucose During the fermentation when glucosesupply in the medium was near depletion, high dose during of glucose wasadded to bring the aqueous fermen- glucose concentration to between 150g/L and tation 250 g/L; no continuous glucose addition was made betweeneach high dose of glucose

FIGS. 1 to 3 are graphs showing the time profile of ONC-T18 cellconcentration (biomass) and total fatty acid production (TFA %), usingdifferent scales of fermentors. Contrary to what has been reported onvarious microorganisms under high carbon concentrations, ONC-T18 wasable to grow very fast under these harsh growth conditions and itsbiomass could increase up to 230 g/L during four to five days offermentation. Final total fat content could reach 70% at all scales offermentation tested, meeting or exceeding those reported in theliterature for single cell oil fermentations.

To investigate whether the ability to grow and produce oil under highcarbon concentrations is a unique trait of the strain ONC-T18 and highlyrelated strains, a representative microalgae oil production strainSchizochytrium sp. ATCC20888 was used to run fermentations in parallelwith ONC-T18. In the first parallel fermentation experiment, bothstrains were grown in 2 L fermentors using medium formula that were thesame as those listed in Table 1, with the initial glucose concentrationbeing 188 g/L in the ONC-T18 fermentor and 193 g/L in the ATCC20888fermentor. No additional glucose was supplied into the ATCC20888fermentor during the fermentation, as no significant consumption of theinitial glucose had occurred. As demonstrated in FIG. 4, the strainSchizochytrium sp. ATCC20888 could not cope with such a high initialconcentration of glucose in the medium and, therefore, had little growthin terms of total biomass.

To confirm that the inability of ATCC20888 to grow under high glucosecondition was not due to the special composition of ONC's algal oilfermentation medium, another set of parallel fermentations were run in 2L fermentors. During this experiment, ONC-T18 was still cultured usingONC's medium formula, while ATCC20888 was cultured using a mediumformula adapted from US patent U.S. Pat. No. 6,607,900 to Bailey et al.,which is incorporated by reference herein in its entirety. Due to timeconstraints, only initial glucose (258 g/L in the ONC-T18 fermentor and211 g/L in the ATCC20888 fermentor) was provided and both fermentationswere terminated before glucose was depleted (FIG. 5). Even with mediumformula that was specifically developed for the strain ATCC20888, highinitial glucose still presented too harsh a condition for the culture togrow significantly.

As has been demonstrated by fermentations above, the major differencebetween traditional fed-batch fermentation and the newly developedfermentation process was the high glucose concentration at the start andalso during the course of the fermentation. The new process starts withabout 200 g/L glucose (as compared to 60 g/L glucose of previousprocesses) and sufficient amounts of other nutrients (e.g., nitrogen inthe form of ammonium sulfate, phosphorus in the form of potassiumphosphate). Once the glucose is depleted or near depletion, as detectedby a quick glucose assay of an off-line sample, another high dose ofglucose is added at once to raise the glucose concentration in thefermentation medium back to around 200 g/L. Therefore, after each timeof the high dose glucose addition, the fermentation was operated underhigh-glucose batch mode. Such cycles of glucose addition and batchoperation is repeated until the oil production reaches the physiologicallimits of the culture, or the growth/production is limited by otherfermentation conditions, such as dissolved oxygen supply, which isdetermined by the design factors of a particular fermentation system.Such a carbon supply strategy greatly simplifies the monitoring andcontrol of algal oil fermentation process. This is evidenced by Table 2showing the validation of this process at 2 L to 10 L. As shown in Table2, with a 4 to 6 day cycle the biomass can reach 200 to 230 g/L with atotal fatty acid content hitting about 70%.

TABLE 2 High glucose multi-batch strategy validation in 2 L to 10 Lcultures. Batch # Batch time Biomass TFA MFA 2011-2L-1 102 h 227 g/L 69%56% 2.22 g/Lh 156 g/L 88 g/L 1.53 g/Lh 0.86 g/Lh 2011-5L-1 136 h 237 g/L69% 68% 1.74 g/Lh 163 g/L 110 g/L 1.20 g/Lh 0.81 g/Lh 2012-10L-3 135 h193 g/L 77% 57% 1.43 g/Lh 149 g/L 86 g/L 1.10 g/Lh 0.63 g/Lh 2012-10L-6119 h 191 g/L 67% 66% 1.61 g/Lh 128 g/L 84 g/L 1.08 g/Lh 0.71 g/Lh2012-10L-4 162 h 227 g/L 70% 60% 1.41 g/Lh 159 g/L 96 g/L 0.98 g/Lh 0.59g/Lh

Another advantage of such high-glucose fermentation is the competitiveedge presented by the high osmotic pressure, which few microorganismsare able to withstand resulting in less contamination. During twofermentations, additional glucose, other than the initial 200 g/Lglucose, was added in a non-sterile form. No contamination was observed.

Example 2 Non-Sterile Fermentation Process for Culturing Microorganismsfor Oil Production

A significant cost to industrial scale fermentation includes thoseassociated with sterilization. The costs include the expense of pressurevessel fermenters and steam-in-place systems as well as operating costsassociated with generating steam. One way in which to reduce these costsis to ferment cultures under non-sterile conditions. However,non-sterile conditions are problematic for most microorganisms due toculture contamination, e.g., by bacteria.

In order to investigate non-sterile conditions in which ONC-T18 andsimilar microorganisms can grow, a medium without yeast extract or soyapeptone was prepared. Table 3 lists the medium components. pH wascontrolled throughout the fermentation to 4.5 using sodium hydroxide(5N). The temperature was not controlled and a glucose feed of 75% wasused during fermentation.

TABLE 3 Initial medium components. No other additions were made to thefermentation except NaOH and phosphoric acid to control pH. IngredientsAmount (per liter) Himedia Yeast Extract 0 g/L Himedia Soya Peptone 0g/L Initial glucose 60 g/L NaCl 9 g/L Ammonium sulfate 20 g/LMonopotassium phosphate 2 g/L Magnesium sulfate 4 g/L Calcium chloride(solution) 0.5 ml/L FeCl3 6H2O (solution) 0.5 ml/L TES (solution) 1.5ml/L Vitamin (solution) 3 ml/L

Air was supplied by a silicone tube with no sparger. The impeller was aLightnin A310 style hydrofoil (axial flow). The open top vessel was abottle with the top removed. The pH was controlled by the Sartorius PHcontrol system on the Biostat B (Sartorius Corporation, Bohemia, N.Y.).There was no temperature control. The rate of fat accumulation over theduration of the fermentation was 0.5 g/L/h and the rate of DHAaccumulation was 0.23 g/L/h. The results are shown in Table 4.

TABLE 4 Final results of open top fermentation. Time (hours) Biomass(g/L) Total Fatty Acids (g/L) DHA (g/L) 161.4 139 79 38

During a 500 L pilot scale run, bacterial contamination was detected atlog hour 8. The contamination was determined by PCR to be in the genusBacillus. The medium components are shown in Table 5.

TABLE 5 Medium components. Ingredients Amount (per liter) Himedia SoyaPeptone 10 g/L Initial glucose 60 g/L NaCl 9 g/L Ammonium sulfate 10 g/LPotassium phosphate 2.2 g/L Potassium phosphate 2.4 g/L Magnesiumsulfate 4 g/L Calcium chloride (solution) 0.5 ml/L FeCl3 6H2O (solution)0.5 ml/L TES (solution) 1.5 ml/L Vitamin (solution) 3 ml/L

The bacteria was counted using a hemocytometer, and its concentrationcalculated to the unit of cell count per ml of media. The bacteriapopulation ceased to increase when the pH was dropped to 3.3. However,even at this low pH, the culture of ONC-T18 contained to grow as shownin FIG. 9. The results are shown in Table 6. It is noted that if thisexperiment were started at pH 3.3 instead of pH 6.5, no bacterialcontamination would have been observed.

TABLE 6 Final results from fermentation assay. Time (hours) Biomass(g/L) Total Fatty Acids (g/L) DHA (g/L) 198 148 103 34

Different pH values were then tested for their effect on themicroorganism, specifically, at pH values of 6.5, 4.5, and 3.2. ONC-T18performed very well under even the very acidic condition. The resultsare shown in FIGS. 7 and 8. Fermentations for FIGS. 7 and 8 were carriedout using high-glucose multi-batch feeding strategy as described inExample 1.

Thus, it is demonstrated herein that ONC-T18 and similar microorganismscan be fermented or grown under high stress conditions, e.g., highglucose (and thus high osmotic pressure) and/or low pH in order toreduce costs of oil production and reduce contamination.

What is claimed is:
 1. A method for producing one or morepolyunsaturated fatty acids, the method comprising: (a) providing amicroorganism capable of producing polyunsaturated fatty acids; (b)providing a medium comprising a high concentration of one or more carbonsources, low pH, or both; and (c) culturing the microorganism in themedium under sufficient conditions to produce the one or morepolyunsaturated fatty acids.
 2. The method of claim 1, wherein themedium has a low pH.
 3. The method of claim 1, wherein the medium has ahigh concentration of one or more carbon sources.
 4. The method of claim1, wherein the medium has a low pH and a high concentration of one ormore carbon sources.
 5. The method of claim 1, wherein the microorganismis ONC-T18.
 6. The method of claim 1, wherein the concentration of theone or more carbon sources is 200 to 300 g/L.
 7. The method of claim 1,wherein the carbon source is selected from the group consisting of fattyacids, lipids, glycerols, triglycerols, carbohydrates, polyols, andamino sugars.
 8. The method of claim 1, wherein the carbon source isglucose.
 9. The method of claim 1, wherein the pH of the medium is 2 to4.5.
 10. The method of claim 9, wherein the pH of the medium is 3 to3.5.
 11. The method of claim 1, further comprising isolating thepolyunsaturated fatty acids, wherein the medium is not sterilized priorto isolation of the polyunsaturated fatty acids.
 12. The method of claim11, wherein sterilization comprises an increase in temperature.
 13. Amethod of reducing contamination of a non-sterile culture comprising oneor more microorganisms, the method comprising culturing themicroorganisms (i) in the presence of a high concentration of one ormore carbon sources, (ii) under conditions of low pH, or (iii) acombination thereof, wherein the culturing reduces contamination of thenon-sterile culture comprising the microorganisms.
 14. The method ofclaim 13, wherein the method comprises culturing the microorganisms inan open vessel.
 15. The method of claim 13, wherein the culturingcomprises culturing the microorganisms in the presence of a highconcentration of one or more carbon sources.
 16. The method of claim 13,wherein the culturing comprises culturing the microorganisms underconditions of low pH.
 17. The method of claim 13, wherein the culturingcomprises culturing the microorganisms in the presence of a highconcentration of one or more carbon sources and under conditions of lowpH.
 18. The method of claim 13, wherein the microorganism is ONC-T18.19. The method of claim 13, wherein the concentration of the one or morecarbon sources is 200 to 300 g/L.
 20. The method of claim 13, whereinthe carbon source is selected from the group consisting of fatty acids,lipids, glycerols, triglycerols, carbohydrates, polyols, and aminosugars.
 21. The method of claim 13, wherein the carbon source isglucose.
 22. The method of claim 13, wherein the pH of the medium is 2to 4.5.
 23. The method of claim 22, wherein the pH of the medium is 3 to3.5.
 24. A method of culturing one or more microorganisms comprising:(a) culturing the microorganisms in a medium comprising a first amountof one or more carbon sources at a first concentration level, whereinthe first concentration level of the one or more carbon sources isgreater than 200 g/L; (b) monitoring a carbon source concentration untilthe carbon source concentration is reduced below the first concentrationlevel; and (c) adding to the medium a second amount of one or morecarbon sources to increase the carbon source concentration to a secondconcentration level, wherein the second concentration level of the oneor more carbon sources is greater than 200 g/L.
 25. The method of claim24, wherein the second amount of the one or more carbon sources is addedto the medium when the carbon source concentration level is reduced to 0to 20 g/L.
 26. The method of claim 24, further comprising, afteraddition of the second amount of the one or more carbon sources, (a)culturing the microorganisms until the carbon source concentration ofthe one or more carbon sources is reduced below the second concentrationlevel and (b) adding to the medium a third amount of one or more carbonsources to increase the carbon source concentration to a thirdconcentration level.
 27. The method of claim 26, wherein the thirdconcentration level of the one or more carbon sources is greater than200 g/L.
 28. The method of claim 26, wherein the third amount of the oneor more carbon sources is added to the medium when the carbon sourceconcentration is reduced to 0 to 20 g/L.
 29. The method of claim 26,further comprising, after addition of the third amount of the one ormore carbon sources, (a) culturing the microorganisms until the carbonsource concentration of the one or more carbon sources is reduced belowthe third concentration level and (b) adding to the medium a fourthamount of one or more carbon sources to increase the carbon sourceconcentration to a fourth concentration level.
 30. The method of claim29, wherein the fourth concentration level of the one or more carbonsources is greater than 200 g/L.
 31. The method of claim 29, wherein thefourth amount of the one or more carbon sources is added to the mediumwhen the carbon source concentration is reduced to 0 to 20 g/L.
 32. Themethod of claim 24, wherein the methods further comprise monitoring thecarbon source concentration one or more times.
 33. The method of claim32, wherein the monitoring comprises measuring dissolved oxygen levels.34. The method of claim 32, wherein monitoring comprises obtaining asample of the medium and determining the carbon source concentration inthe sample.
 35. The method of claim 24, wherein the microorganism isONC-T18.
 36. The method of claim 24, wherein the microorganisms arecapable of producing one or more polyunsaturated fatty acids.
 37. Themethod of claim 36, wherein the method further comprises isolating thepolyunsaturated fatty acids produced by the microorganisms.
 38. Themethod of claim 34, wherein the carbon source is selected from the groupconsisting of fatty acids, lipids, glycerols, triglycerols,carbohydrates, polyols, and amino sugars.
 39. The method of claim 34,wherein the carbon source is glucose.
 40. The method of claim 29,wherein the one or more carbon sources in the first, second, third, andfourth amounts are the same.